Considering the large number of complex pathways involved in the fruit ripening process, and despite construction and screening of many tomato fruit cDNA libraries in the past decade, surprisingly few fruit- or ripening-specific genes and their functions have been identified. In order to address this specific area a new cDNA library was constructed (pERT clone series, Early Ripening Tomato) from the pericarp of a very early ripening stage of wild-type tomato fruit and differentially screened against mRNA obtained from the pericarp of ripening inhibitor (rin) mutant fruit of a similar developmental stage.
The rin mutation, first reported in 1968, is recessive, maps to chromosome 5 and is closely linked to the macrocalyx locus. It has pleiotropic effects on ripening, resulting in an extremely retarded ripening phenotype. Fruit demonstrate an increased resistance to many common post harvest pathogens and have been maintained for years without further signs of normal ripening or deterioration. Following extensive storage of fruit seeds may germinate and grow precociously within the fruit. Other aspects of plant growth and early fruit development appear unaffected by the rin mutation. However, the rin fruit fail to attain a normal level of pigmentation as a result of decreased accumulation of carotenoids, particularly lycopene, and there is a decreased rate of chlorophyll loss so that rin fruit remain green when wild-type fruit are fully red. The rin fruit eventually "ripen" to a lemon yellow colour after several weeks. These mutant fruit also fail to achieve normal flavour or aroma which has been correlated with the reduced production of a number of aromatic compounds.
Despite these considerable shortfalls the rin mutation is used in the heterozygous state in commercial tomato production, as in such a genetic state the deficiencies of the homozygous rin mutation regarding fruit quality are at least partially overcome. The major benefit of rin heterozygous tomatoes is the maintained firmness which gives the tomato improved handling characteristics particularly for fresh market applications.
Examination of total proteins of wild-type and rin pericarp reveals differences during ripening, some proteins being more abundant and others reduced in the mutant fruit as compared to wild-type. In vitro translation profiles suggest that such changes are the result of altered gene expression in the mutant fruit. Subsequent analysis with ripening-related cDNA clones showed altered patterns of accumulation of several mRNAs in the mutant fruit, suggesting that the rin mutation effects expression of many ripening-related genes.
At the onset of ripening, rin fruit do not show the autocatalytic rise in ethylene evolution characteristic of normal tomato fruit and ripen essentially as non-climacteric fruit. Therefore, it has been suggested that the rin mutation may affect ethylene receptors end thus lead to the retarded ripening phenotype. However, there is evidence that rin fruit are able to perceive ethylene but ethylene alone is unable to reverse the mutant phenotype. Application of high levels of exogenous ethylene does induce red pigmentation in rin fruit and leads to an increase in respiration rate but fails to induce autocatalytic ethylene production. Ethylene treatment also restores accumulation of some ripening-related mRNAs which are substantially reduced in the mutant fruits namely those homologous to PTOM 5 (phytoene synthase), pTOM 13 (ethylene-forming enzyme), pTOM 99 (now known to be encoded by the ethylene responsive gene E8) and E4, but fails to significantly increase accumulation of polygalacturonase (pTOM 6) mRNA.
rin fruit, as a non-climacteric background, have been used to examine transcriptional activation and accumulation of a number of ethylene-responsive genes. These experiments indicate that rin effects both transcriptional and post-transcriptional events. Transcription of both polygalacturonase and E4 genes, and thus subsequent mRNA accumulation, are effectively abolished in rin fruit. In contrast, transcription of the E8 (pTOM 99) and J49 genes is reduced and their homologous mRNAs accumulate at a reduced level. In the case of the E17 gene, the rin mutation appears to have no effect on transcription, but mRNA accumulation is severely impaired.
In view of the extreme nature of the rin ripening mutant and its diverse effects at the molecular level, where it affects the expression of many ripening-related genes, it is not an ideal breeding tool. However due to the nature of the mutation, we decided to use rin fruit mRNN as a probe to isolate fruit ripening-related cDNAs. A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at various levels in fruit of the tomato ripening mutant, ripening inhibitor (rin). We have now isolated, characterized and sequenced a series of new cDNA clones whose homologous mRNAs show altered patterns of expression in rin fruit during ripening.